Novel transmural preparation from human failing hearts to study phosphoproteins

Phosphorylation of proteins involved in the regulation of contractile strength and relaxation speed are critical to cardiac output regulation. This phosphorylation happens very fast; in the fight-fright-flight response, phosphorylation occurs in mere seconds and can dissipate in tens of seconds. Likewise, in heart-rate dependent regulation of contractility, phosphoproteins are also prominently involved. In order to study phosphorylation levels in human tissue, tissue needs to be collected ideally under a well-controlled physiological state. Langendorff non-working and working heart preparations of small rodents have long been used to study contraction and kinetic parameters, analyze the immediate effects of compounds, and conduct subsequent molecular analyses in a whole beating heart model. However, obtaining nor maintaining a human heart in intact condition to allow for a working heart preparation or Langendorff mode experiments is not readily feasible. Hence, our goal was to develop a protocol that allows for collection of sufficient amounts of tissue (grams) under controlled physiological homeostases. From explanted failing and non-failing hearts, transmural sections weighing approximately 15-20 g are perfused through the in-situ coronary vasculature and stimulated to contract via platinum electrodes. We are able to freeze-clamp a small portion of this wedge that is in contractile homeostasis and thereafter establish a new physiological homeostasis (i.e. at a higher heart rate or in presence of beta-stimulation) and once again freeze-clamp a small portion of this wedge. For transmural sections of both the right and left ventricular free wall, we have shown its viability for both non-failing and end-stage failing human hearts. We are currently applying this method in a preliminary study of phosphoproteins potentially underlying the deficit of frequency-dependent acceleration of relaxation in human end-stage heart failure.